I'm encountering problems with amplifying the same gene when I use different primers -
F1: GGC TAG TCT AGA ATG (TGT ACG CGG TTC GTT TAT CTG GAT C)
R1: CGA GTA GAT CTC (TAG AGC CCC GCG AAT TCA AAC G)
F2: GGA GCT CAT ATG (TGT ACG CGG TTC GTT TAT C)
R2: GTA ATG AAG CTT (CTA GAG CCC CGC GAA TTC)
The nucleotides inside () form the sequence that is identical to the gene sequence. The primers are almost the same, except that I've changed the restriction sites. I get good amplification with the first set of primers (annealing temp 56 C), but there is completely no product with the second set at annealing temperatures from 45 to 57. How should I proceed? The genomic dna and other components are all same except the primers. Is there some aspect of primer design that I've overlooked?
Hi, and you are sure that the melting temperature (Tm) of the primers used are not more than 5°C different from each other?! Cheers, Nadine
make sure your new primers are have a same orientation of 5' and 3' ends with the F1 and R1 primers. You have remove several base in the 3'end, I think the 3'end of the new primer is not anneal with your template. So, the amplification does not occur.
Dear Avinash,
According to my analysis (BLAST, Sequence matching) your extended primer of R2 (GTAATGAAGCTT) is matching somewhere at your target sequence (which should not be). So, try to change it (suggestion).
All the best